Stage-specific gut proteinases of the cotton stainer bug Dysdercus peruvianus: role in the release of entomotoxic peptides from Canavalia ensiformis urease.
Canavalia ensiformis ureases are toxic to insects of different orders. The entomotoxicity of urease is due to a 10 kDa internal peptide released by proteinases in the insect digestive tract. We previously observed that, given orally, urease is toxic to nymphs of Dysdercus peruvianus, but does not affect adults. Here we characterized the major proteolytic activities of D. peruvianus midgut homogenates and investigated their in vitro-catalyzed release of the 10 kDa entomotoxic peptide from urease. Cysteine, aspartic and metalloproteinases are present in both homogenates. Variations in optimal pH and susceptibility to inhibitors indicated differences in the enzyme profiles in the two developmental stages. Only nymph homogenates released approximately 10 kDa fragment(s) from urease, recognized by antibodies against the entomotoxic peptide. Fluorogenic substrates containing urease partial sequences flanking the N-terminal or the C-terminal portion of the entomotoxic peptide were efficiently cleaved by homogenates from nymphs, but much more slowly by the adult homogenate. Different classes of enzymes in the homogenates cleaved both substrates suggesting that in vivo the release of the entomotoxic peptide results from the concerted action of at least two different proteinases. Our findings support the view that a differential processing of ingested urease by the insects explains at least in part the lack of toxicity in adults.
Piovesan
Angela R
AR
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Building 43421, CEP 91501-970 Porto Alegre, Rio Grande do Sul, Brazil.
Stanisçuaski
Fernanda
F
Marco-Salvadori
Juliana
J
Real-Guerra
Rafael
R
Defferrari
Marina S
MS
Carlini
Célia R
CR
D000818:Animals; D032449:Canavalia / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002853:Chromatography, Liquid; D020062:Heteroptera / Q000201:enzymology* / Q000254:growth & development; D006868:Hydrolysis; D019476:Insect Proteins / Q000378:metabolism / Q000502:physiology*; D009758:Nymph / Q000201:enzymology / Q000254:growth & development; D010447:Peptide Hydrolases / Q000378:metabolism / Q000502:physiology*; D021241:Spectrometry, Mass, Electrospray Ionization; D014118:Toxins, Biological / Q000378:metabolism*; D014510:Urease / Q000378:metabolism*
D002364:Caseins; D019476:Insect Proteins; D014118:Toxins, Biological; C026018:azocasein; D010447:Peptide Hydrolases; D014510:Urease